During the 1980's, compounds comprising a series of structurally related complex polyketides were investigated for there antineoplastic properties. The compounds, named bryostatins, were isolated from the marine bryozoan, Bugula neritina, a sessile marine invertebrate that filters plankton and other particles from the water column. One of these compounds, bryostatin 1, was shown to be particularly effective against leukemia. Since the time of the initial finding of bryostatin 1, numerous other bryostatins have been discovered, pharmacologically tested, and shown to also significantly inhibit the proliferation of cancer cells in vitro and in in vivo animal studies. For example, U.S. Pat. No. 4,560,774 discloses the compositions and structures of bryostatins 1 to 3; U.S. Pat. No. 4,611,066 discloses the compositions and structures of bryostatins 4 to 8; and U.S. Pat. No. 4,833,257 discloses the compositions and structures of bryostatins 9 to 13. In all, nineteen bryostatin compositions and structures are known and have been identified.
The most prominent source of bryostatins is the organism Bugula neritina, a marine bryozoan; some bryostatins have been isolated from other marine invertebrates but these samples were reported to be contaminated with colonies of Bugula neritina. Bugula neritina is a common fouling organism found in virtually all temperate regions of the world's oceans. It occurs as bushy colonies, branching biserial to about 10 cm high. Typically a brown-purplish color, the organisms may be found attached to many surfaces in coastal and estuarine environments, including rock jetties, floating docks, and on the bottoms of ships and boats. More recently, man-made aquacultural environments and man-made oceanic aquacultural environments have been studied for growing Bugula neritina under more controlled conditions. The focus of these studies has been to attempt to produce a sufficient, easily harvestable biomass of Bugula neritina from which commercially viable quantities of bryostatins could be isolated.
However, the reported yields of bryostatins from Bugula neritina are extremely low. For instance, bryostatins are typically reported to occur in concentrations on the order of 10−6 to 10−8 percent Bugula neritina wet mass. Existing methods for the recovery of bryostatins from Bugula neritina requires the destructive harvesting and processing of the organism. To obtain usable quantities of bryostatins, an enormous biomass of Bugula neritina must be harvested and processed. A couple of methods have been proposed and tested for the harvesting of the Bugula neritina for bryostatin acquisition. One proposed harvesting method involves the collection of wild Bugula neritina growing in natural coastal environments. For example, to acquire sufficient quantities of bryostatin 1 just to conduct Phase I clinical trials, the National Cancer Institute collected and processed 12,712 kilograms of Bugula neritina to obtained approximately 18 grams of bryostatin. However, concerns about environmental impacts resulting from over-collecting and the possible decimation of local populations of Bugula neritina have led to the desire to develop alternative methods for growing and harvesting the organism. In response to this need, man-made aquacultural environments have been developed to grow Bugula neritina for harvesting and processing for bryostatins. The man-made aquacultural environments appear to be promising and offer some hope that large amounts of Bugula neritina organisms may be grown to meet the eventual biomedical and other research and commercial demands for bryostatins.
For instance, CalBioMarine, Inc., a California corporation, has developed methods for initiating the growth of Bugula neritina organisms on plastic or polymer plates in a controlled, man-made aquatic environment. After establishing seed populations of Bugula neritina on artificial, plastic plates, these substrates with growing Bugula neritina are transferred to an ocean environment where Bugula neritina growth is vigorous. These plates with attached Bugula neritina may be removed from the ocean for the harvesting and subsequent chemical processing of the Bugula neritina. The plates may be cleaned and reused for the next growth cycle by repeating the process described above. In addition to being expensive, this form of growing and harvesting Bugula neritina is also susceptible to failure at several points in the process. For example, if the Bugula neritina used to seed the plates do not initially grow well, an entire season's crop may be lost. To minimize some of these potential problems, other growth and harvesting strategies have been proposed for the aquaculture-base growth of Bugula neritina. For example, “clip-harvesting” of Bugula neritina growing on plates suspended in ocean waters has been proposed as an alternative harvesting method. Using clip harvesting, the entire Bugula neritina organism is not destroyed. This allows the Bugula neritina to re-grow after a portion is clipped from each colony. It is hypothesized that the clip-harvesting method may eliminate the costly process of seeding the plates with new Bugula neritina each year, as well as the need to monitor and control the growth of the Bugula neritina on the plates in a man-made environment until such time that they may be transferred to the ocean for further growth. However, relative to a completely destructive harvest, larger standing stock of Bugula neritina must be maintained for clip harvesting to acquire the same biomass of Bugula neritina to attain a given amount of bryostatins. Further, the costs of clip harvesting, while possibly lower than the destructive harvesting, are still high.
One major problem associated with acquiring bryostatins from Bugula neritina is the exceedingly low levels of bryostatins in Bugula neritina—typically in the range of 10−6 to 10−8 percent wet mass of the animal. Using current processing techniques, large quantities of the Bugula neritina must be harvested and processed to extract very small concentrations of bryostatins from the organism, which substantially increases the costs of removing and purifying brysotatins from harvested Bugula neritina. During processing, the bryostatins are extracted from the Bugula neritina along with numerous other compounds produced by the animal. The bryostatins must then be purified from the complex mixture of chemicals in this extract. The purification of bryostatins from the Bugula neritina extract requires a great number of man-hours, the use of large amounts of chemicals, and expensive processing equipment, such as preparative-scale chromatography systems. Solvents typically used in the processing steps create health and safety hazards, pose environmental concerns, and increase the costs of processing. In addition, large amounts of solvent treated Bugula neritina organisms must be disposed of following processing. The overall costs of processing Bugula neritina to obtain bryostatins are very high, thereby making the acquisition of commercial quantities of bryostatins from Bugula neritina uneconomical while limiting the amount of bryostatin produced annually.
A cost-effective and commercially scalable synthesis of bryostatins has not yet been developed, which has further hindered the biomedical development and commercialization of the bryostatins. Although numerous research groups are currently working on developing methods for the efficient, large-scale and economical synthesis of bryostatins, the structural complexity of the bryostatins has prevented the development of an economically viable synthesis and thus the commercial development of the bryostatins.
Given the major impediments discussed above for producing commercially viable quantities of bryostatin-based cancer drugs and valuable molecular probes, there exists a tremendous need and desire to develop new methods for obtaining bryostatins that are economically feasible. Furthermore, the development of new bryostatin compounds and structures is also desirable.